Current studies indicate that development of cervical cancer is strongly linked to the DNA integration of several subtypes of the human papillomavirus (HPV). The Pap test has helped in the early detection of precursor lesions; however, results are inconclusive when low grade lesions are detected. In these cases, a solution based hybridization method can be used to screen for the presence and viral burden of common HPV subtypes associated with cervical cancer. The presence of HPV, however, is not a useful indicator of subsequent development of cervical cancer. A test which can determine the occurrence of viral integration is a better indicator of disease. Current in situ hybridization methods have limited success in distinguishing integrated virus from episomal virus. Analysis is difficult because both episomal and integrated virus often coexist, and the "diffuse" pattern indicating episomal virus often masks the "dot" pattern indicating integrated virus. By combining single cell gel microdrop (GMD) encapsulation technology, in situ hybridization and nuclear fractionation techniques, this Phase I proposal aims to develop an assay which will unambiguously distinguish integrated virus from episomal virus. Use of GMD technology will also reduce cell loss, facilitating rare cell detection. PROPOSED COMMERCIAL APPLICATIONS: The assay will permit early detection of cervical cancer and highly specific assessment of viral pathogenic mechanisms directly linked to cervical cancer, facilitating early treatment and reducing mortality rates.